Haematococcus pluvialis, a unicellular green microalga that produces a secondary metabolite under stress conditions, bears one of the most potent antioxidants, namely xanthophyll astaxanthin. The aim of our study was to determine the content of astaxanthin and its esterified forms using three different solvents—methyl tert-butyl ether (MTBE), hexane isopropanol (HEX -IPA) and acetone (ACE)—and to identify them by using high performance liquid chromatography coupled with diode array detection and the quadrupole time-of-flight mass spectrometry (HPLC-DAD and LC-QTOF-MS) technique. We identified eleven astaxanthin monoesters, which accounted for 78.8% of the total astaxanthin pool, six astaxanthin diesters (20.5% of total), while free astaxanthin represented the smallest fraction (0.7%). Astaxanthin monoesters (C16:2, C16:1, C16:0), which were the major bioactive compounds in the H. pluvialis samples studied, ranged from 10.2 to 11.8 mg g⁻¹ DW. Astaxanthin diesters (C18:4/C18:3, C18:1/C18:3) were detected in the range between 2.3 and 2.6 mg g⁻¹ DW. All three solvents were found to be effective for extraction, but MTBE and hexane-isopropanol extracted the greatest amount of free bioactive astaxanthin. Furthermore, MTBE extracted more low-chain astaxanthin monoesters (C16), and hexane-isopropanol extracted more long-chain monoesters (C18 and above) and more diesters. We can conclude that MTBE is the solvent of choice for the extraction of monoesters and hexane-isopropanol for diesters.

H. pluvialis is a unicellular freshwater alga containing many bioactive compounds, especially carotenoids, which are the strongest antioxidants among the pigments. This study evaluates the composition and content of carotenoids and other pigments in both stages of algae life cycle, especially in the green vegetative stage, less studied in comparison to the red stage. To determine the composition and content of carotenoids, a combination of HPLC-DAD and LC-QTOF-MS was used. The content of carotenoids in the green vegetative stage was significantly lower than in the red vegetative stage. In the green vegetative stage, 16 different carotenoids and other pigments were identified. Among the total 8.86 mg g⁻¹ DW of pigments, 5.24 mg g⁻¹ DW or 59% of them were chlorophyll a with its derivatives, and 3.62 mg g⁻¹ DW or 41% of them were free carotenoids. After the transition from the green to the red stage, the carotenoid composition was replaced by secondary carotenoids, astaxanthin and its esters, which predominated in the whole carotenoid composition. In addition to free astaxanthin, 12 astaxanthin monoesters, 6 diesters and 13 other carotenoids were determined. The majority of 37.86 mg g⁻¹ DW pigments were monoesters. They represented 82% of all pigments, and their content was about 5 times higher than both, diesters (5.91 mg g⁻¹ DW or 12% of all) and free carotenoids (2.4 mg g⁻¹ DW or 6% of all). The results of the study contribute to the data on the overall pigment composition and content of H. pluvialis algae and provide the basis for further improvement of cultivation of the H. pluvialis algae.

Chlorogenic acid is one of the most prominent bioactive phenolic acids with great pharmacological, cosmetic and nutritional value. The potential of Berula erecta in tissue culture was investigated for the production of chlorogenic acid and its elicitation combined with light of different wavelengths and low temperature. The content of chlorogenic acid in the samples was determined by HPLC-UV, while the content of total phenolic compounds and the antioxidant activity of their ethanol extracts were evaluated spectrophotometrically. The highest fresh and dry biomasses were obtained in plants grown at 23 °C. This is the first study in which chlorogenic acid has been identified and quantified in Berula erecta. The highest chlorogenic acid content was 4.049 mg/g DW. It was determined in a culture grown for 28 days after the beginning of the experiment at 12 °C and under blue light. The latter also contained the highest content of total phenolic compounds, and its extracts showed the highest antioxidant activity. Berula erecta could, potentially, be suitable for the in vitro production of chlorogenic acid, although many other studies should be conducted before implementation on an industrial scale.

The monotypic Hladnikia pastinacifolia Rchb. is a narrow endemic species, with an extremely small distribution area in Slovenia, prone to any kind of threat that could lead to species extinction. Tissue culture techniques are proposed as a conservation measure for rapid propagation and ex-situ conservation. Tissue culture was initiated from seeds and juvenile plants obtained from natural sites on a solid Murashige and Skoog (MS) medium, with and without growth regulators. We tested various combinations and concentrations of growth regulators, and the best proliferation of axillary shoots, on average 14, was obtained on MS medium with 5 μM BAP and 3 μM IBA and 3% sucrose. Rooting was achieved after transferral of the shoots to an MS medium with 2 μM IBA and 3% sucrose. The rooted plants were acclimatized on a mixture of limestone sand, potting soil and vermiculite in a ratio of 10:2:2, with pH in the range of 7.5–8.0. In vitro propagation methods provide an important opportunity for the propagation and preservation of H. pastinacifolia by rapidly increasing the number of plants, without disturbing the wild population.